Stem cells, both embryonic and somatic, hold great potential for tissue and function restoration in human diseases. However, our lack of understanding regarding the precise process by which stem cells differentiate towards a particular phenotype or even the optimal source of stem cells with the greatest therapeutic potential for a particular disease reflects gaps in our knowledge of the fundamental stem cell biology and remains an obstacle to effective clinical translation. The search for "stemness" genes has suggested that gene expression alone is not sufficient to insure or define either plasticity or lineage specification. We hypothesize (a) that characterization of human stem cells by epigenetic processes will provide an underlying mechanism for the pluripotency of undifferentiated human embryonic stem cells (hESCs) and the differentiation cascades of their progeny; (b) that epigenetic marks, as established across development by the dynamics of chromatin remodeling, can be used to define potency, plasticity, and lineage-commitment along the continuum of human stem cell development; and (c) that such marks can be used to judge the therapeutic potential of a cell. The goal of this research proposal is to study the epigenetic controls of the hESC (NIH registry code: WA01, WA07, and WA09) as it differentiates towards a human neural stem cell (hNSC) and then a dopaminergic (DA) phenotype. Having established in our lab strategies for differentiating pluripotent hESCs towards becoming multipotent hNSCs and then towards DA neurons under defined culture conditions, and having generated a number of hNSC lines which can also be directed towards a DA phenotype, I will characterize the differentiation process by examining the progression of chromatin states and identify epigenetic landmarks. The profile of epigenetic marks in hESC differentiation will be compared to that of the CNS-derived hNSCs and their differentiated DA neurons. The identified epigenetic landmarks will be used to define and compare the plasticity and potential of human stem cells. These characteristics will be further affirmed by using an in vivo bioassay (a mouse model of DA dysfunction in the aged brain) to test and predict the therapeutic potential of a given stem cell state. The mentorship I will receive in the Snyder lab while pursuing the goals of this proposal will be significant for developing my future career as an independent human stem cell investigator.